Multi-Omics Analysis of Gene and Protein Candidates Possibly Related to Tetrodotoxin Accumulation in the Skin of Takifugu flavidus.

Pufferfish is increasingly regarded by many as a delicacy. However, the tetrodotoxin (TTX) that accumulates in its body can be lethal upon consumption by humans. TTX is known to mainly accumulate in pufferfish skin, but the accumulation mechanisms are poorly understood. In this study, we aimed to explore the possible mechanism of TTX accumulation in the skin of the pufferfish Takifugu flavidus following treatment with TTX. Through liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, we detected 37.3% of toxin accumulated in the skin at the end of the rearing period (168 h). Transcriptome and proteome analyses revealed the mechanism and pathways of TTX accumulation in the skin of T. flavidus in detail. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes analyses strongly suggest that cardiac muscle contraction and adrenergic signaling in cardiomyocyte pathways play an important role in TTX accumulation. Moreover, some upregulated and downregulated genes, which were determined via RNA-Seq, were verified with qPCR analysis. This study is the first to use multi-omics profiling data to identify novel regulatory network mechanisms of TTX accumulation in the skin of pufferfish.

A Microencapsulation Method for Delivering Tetrodotoxin to Bivalves to Investigate Uptake and Accumulation.

Most marine biotoxins are produced by microalgae. The neurotoxin tetrodotoxin (TTX) has been reported in many seafood species worldwide but its source is unknown, making accumulation and depuration studies in shellfish difficult. Tetrodotoxin is a water-soluble toxin and cannot be directly ingested by shellfish. In the present study, a method was developed which involved binding TTX to solid particles of humic acid and encapsulating them in agar-gelatin capsules. A controlled quantity of TTX-containing microcapsules (size range 20-280 µm) was fed to Paphies australis, a bivalve known to accumulate TTX in the wild. The TTX-containing microcapsules were fed to P. australis every second day for 13 days. Ten P. australis (including five controls fed non-toxic microalgae) were harvested after 7 days and ten after 13 days. Paphies australis accumulated TTX, reaching concentrations of up to 103 µg kg-1 by day 13, exceeding the European Food Safety Authority recommended concentration of 44 µg kg-1 in shellfish. This novel method will allow future studies to explore the effects, accumulation and depuration rates of TTX in different animals and document how it is transferred through food webs.

Safety, Tolerability, Pharmacokinetics, and Concentration-QTc Analysis of Tetrodotoxin: A Randomized, Dose Escalation Study in Healthy Adults.

Tetrodotoxin (TTX) is a highly specific voltage-gated sodium channel (VGSC) blocker in clinical evaluation as a peripheral-acting analgesic for chronic pain. This study presents the first published results of the safety including cardiac liability of TTX at therapeutic-relevant concentrations in twenty-five healthy adults. Randomized, double-blind, placebo-, and positive- (moxifloxacin) controlled study evaluated single ascending doses of 15 µg, 30 µg, and 45 µg TTX over 3 periods with a 7-day washout between each period. Subcutaneous injections of TTX were readily absorbed, reaching maximum plasma concentration (Cmax) within 1.5 h. Both extent of exposure (AUC) and Cmax increased in proportion to dose. No QT prolongation was identified by concentration-QTc analysis and the upper bounds of the two-sided 90% confidence interval of predicted maximum baseline and placebo corrected QTcF (ΔΔQTcF) value did not exceed 10 ms for all tetrodotoxin doses, thereby meeting the criteria of a negative QT study. Safety assessments showed no clinically relevant changes with values similar between all groups and no subject withdrawing due to adverse events. Paresthesia, oral-paresthesia, headache, dizziness, nausea, and myalgia were the most common TEAEs (overall occurrence ≥5%) in the TTX treatment groups. TTX doses investigated in this study are safe, well-tolerated, and lack proarrhythmic proclivity.

Oral Chronic Toxicity of the Safe Tetrodotoxin Dose Proposed by the European Food Safety Authority and Its Additive Effect with Saxitoxin.

Tetrodotoxin (TTX) is a potent natural toxin causative of human food intoxications that shares its mechanism of action with the paralytic shellfish toxin saxitoxin (STX). Both toxins act as potent blockers of voltage-gated sodium channels. Although human intoxications by TTX were initially described in Japan, nowadays increasing concern about the regulation of this toxin in Europe has emerged due to its detection in fish and mollusks captured in European waters. Currently, TTX is only regularly monitored in Dutch fishery products. However, the European Food Safety Authority (EFSA) has established a safety level of 44 µg/kg TTX as the amount of toxin that did not cause adverse effects in humans. This level was extrapolated considering initial data on its acute oral toxicity and EFSA remarked the need for chronic toxicity studies to further reduce the uncertainty of future toxin regulations. Thus, in this work, we evaluated the oral chronic toxicity of TTX using the safety levels initially recommended by EFSA in order to exclude potential human health risks associated with the worldwide expanding presence of TTX. Using internationally recommended guidelines for the assessment of oral chronic toxicity, the data provided here support the proposed safety level for TTX as low enough to prevent human adverse effects of TTX even after chronic daily exposure to the toxin. However, the combination of TTX with STX at doses above the maximal exposure level of 5.3 µg/kg body weight derived by EFSA increased the lethality of TTX, thus confirming that both TTX and paralytic shellfish toxins should be taken into account to assess human health risks.

A neural circadian signal essential for ovulation is generated in the suprachiasmatic nucleus during each stage of the oestrous cycle.

NEW FINDINGS: What is the central question of this study? Is the suprachiasmatic nucleus the structure that generates the neural circadian signals that occur during every stage of the oestrous cycle, not only pro-oestrus, and are these signals essential for proper regulation of ovulation? What is the main finding and its importance? Transient inhibition of Na+ -dependent action potentials in the suprachiasmatic nucleus by tetrodotoxin microinjection at 14.00 h inhibits ovulation irrespective of the stage of the oestrous cycle at which the procedure is performed. Microinjection of saline solution into the suprachiasmatic nucleus has a disruptive effect on ovulation that depends on the stage of the cycle at which it is administered. ABSTRACT: Reproduction is a highly timed process that depends on both the reproductive and circadian systems. The core oscillator of the latter resides at the suprachiasmatic nuclei (SCN) and it is pivotal for the regulation of the pro-oestrus pre-ovulatory surge of gonadotropins in females. There is evidence to suggest that this system may be involved in the regulation of neuroendocrine events that are essential for ovulation and that occur prior to pro-oestrus. We explored this possibility by transiently inactivating the SCN. Female rats were implanted with guide cannulas aimed at the SCN. After recovery of the oestrous cycle, animals were injected with tetrodotoxin (TTX), artificial cerebrospinal fluid (ACSF) or saline solution while freely moving. Injections were performed at 14.00 h of each stage of the oestrous cycle. Animals were killed on the next predicted oestrus day, the number of ova shed was counted and intact rats at oestrus stage were used as absolute control. ACSF did not modify ovulation. Saline solution blocked ovulation in oestrus- and dioestrus-injected rats. Irrespectively of the stage of the oestrous cycle, TTX blocked ovulation. These results lead us to suggest that a neural circadian signal, pivotal for triggering the gonadotropin pre-ovulatory surge, arises from the SCN during the critical window of pro-oestrus. We also suggest that a similar signal, needed for the regulation of other events that are indispensable for proper regulation of ovulation, is also generated in this nucleus during the other stages of the cycle at a similar time.

Combination Formulation of Tetrodotoxin and Lidocaine as a Potential Therapy for Severe Arrhythmias.

Severe arrhythmias-such as ventricular arrhythmias-can be fatal, but treatment options are limited. The effects of a combined formulation of tetrodotoxin (TTX) and lidocaine (LID) on severe arrhythmias were studied. Patch clamp recording data showed that the combination of LID and TTX had a stronger inhibitory effect on voltage-gated sodium channel 1.5 (Nav1.5) than that of either TTX or LID alone. LID + TTX formulations were prepared with optimal stability containing 1 µg of TTX, 5 mg of LID, 6 mg of mannitol, and 4 mg of dextran-40 and then freeze dried. This formulation significantly delayed the onset and shortened the duration of arrhythmia induced by aconitine in rats. Arrhythmia-originated death was avoided by the combined formulation, with a decrease in the mortality rate from 64% to 0%. The data also suggests that the anti-arrhythmic effect of the combination was greater than that of either TTX or LID alone. This paper offers new approaches to develop effective medications against arrhythmias.

The Duration of Nerve Block from Local Anesthetic Formulations in Male and Female Rats.

PURPOSE: It is unknown whether there are sex differences in response to free or encapsulated local anesthetics. METHODS: We examined nerve block duration and toxicity following peripheral nerve blockade in male and female rats. We studied the local anesthetic bupivacaine (free or encapsulated) as well as tetrodotoxin, which acts on a different site of the same voltage-gated channel. RESULTS: Sensory nerve blockade was 158.5 [139-190] minutes (median [interquartile range]) (males) compared to 173 [134-171] minutes (females) (p = 0.702) following bupivacaine injection, N = 8 male, 8 female. Motor nerve blockade was 157 [141-171] minutes (males) compared to 172 [146-320] minutes (females) (p = 0.2786). Micellar bupivacaine (N = 8 male, 8 female) resulted in sensory nerve blockade of 266 [227-320] minutes (males) compared to 285 [239-344] minutes (females) (p = 0.6427). Motor nerve blockade was 264 [251-264] minutes (males) compared to 287 [262-287] minutes (females) (p = 0.3823). Liposomal bupivacaine (N = 8 male, 8 female) resulted in sensory nerve blockade of 240 [207-277] minutes (males) compared to 289 [204-348] minutes (females) (p = 0.1654). Motor nerve blockade was 266 [237-372] minutes (males) compared to 317 [251-356] minutes (females) (p = 0.6671). Following tetrodotoxin injection (N = 12 male,12 female) sensory nerve blockade was 54.8 [5-117] minutes (males) compared to 54 [14-71] minutes (females) (p = 0.6422). Motor nerve blockade was 72 [40-112] minutes (males) compared to 64 [32-143] minutes (females) (p = 0.971). CONCLUSIONS: We found no statistically significant sex differences associated with the formulations tested. In both sexes, durations of nerve block were similar between micellar and liposomal bupivacaine formulations, despite the micellar formulation containing less drug.

Prolonged Duration Local Anesthesia by Combined Delivery of Capsaicin- and Tetrodotoxin-Loaded Liposomes.

BACKGROUND: Capsaicin, the active component of chili peppers, can produce sensory-selective peripheral nerve blockade. Coadministration of capsaicin and tetrodotoxin, a site-1 sodium channel blocker, can achieve a synergistic effect on duration of nerve blocks. However, capsaicin can be neurotoxic, and tetrodotoxin can cause systemic toxicity. We evaluated whether codelivery of capsaicin and tetrodotoxin liposomes can achieve prolonged local anesthesia without local or systemic toxicity. METHODS: Capsaicin- and tetrodotoxin-loaded liposomes were developed. Male Sprague-Dawley rats were injected at the sciatic nerve with free capsaicin, capsaicin liposomes, free tetrodotoxin, tetrodotoxin liposomes, and blank liposomes, singly or in combination. Sensory and motor nerve blocks were assessed by a modified hotplate test and a weight-bearing test, respectively. Local toxicity was assessed by histologic scoring of tissues at the injection sites and transmission electron microscopic examination of the sciatic nerves. Systemic toxicity was assessed by rates of contralateral nerve deficits and/or mortality. RESULTS: The combination of capsaicin liposomes and tetrodotoxin liposomes achieved a mean duration of sensory block of 18.2 hours (3.8 hours) [mean (SD)], far longer than that from capsaicin liposomes [0.4 hours (0.5 hours)] (P < .001) or tetrodotoxin liposomes [0.4 hours (0.7 hours)] (P < .001) given separately with or without the second drug in free solution. This combination caused minimal myotoxicity and muscle inflammation, and there were no changes in the percentage or diameter of unmyelinated axons. There was no systemic toxicity. CONCLUSIONS: The combination of encapsulated tetrodotoxin and capsaicin achieved marked prolongation of nerve block. This combination did not cause detectable local or systemic toxicity. Capsaicin may be useful for its synergistic effects on other formulations even when used in very small, safe quantities.

Polymer-tetrodotoxin conjugates to induce prolonged duration local anesthesia with minimal toxicity.

There is clinical and scientific interest in developing local anesthetics with prolonged durations of effect from single injections. The need for such is highlighted by the current opioid epidemic. Site 1 sodium channel blockers such as tetrodotoxin (TTX) are extremely potent, and can provide very long nerve blocks but the duration is limited by the associated systemic toxicity. Here we report a system where slow release of TTX conjugated to a biocompatible and biodegradable polymer, poly(triol dicarboxylic acid)-co-poly(ethylene glycol) (TDP), is achieved by hydrolysis of ester linkages. Nerve block by the released TTX is enhanced by administration in a carrier with chemical permeation enhancer (CPE) properties. TTX release can be adjusted by tuning the hydrophilicity of the TDP polymer backbone. In vivo, 1.0-80.0 µg of TTX released from these polymers produced a range of durations of nerve block, from several hours to 3 days, with minimal systemic or local toxicity.

In vitro validation of in silico identified inhibitory interactions.

BACKGROUND: Understanding how neuronal signals propagate in local network is an important step in understanding information processing. As a result, spike trains recorded with multi-electrode arrays (MEAs) have been widely used to study the function of neural networks. Studying the dynamics of neuronal networks requires the identification of both excitatory and inhibitory connections. The detection of excitatory relationships can robustly be inferred by characterizing the statistical relationships of neural spike trains. However, the identification of inhibitory relationships is more difficult: distinguishing endogenous low firing rates from active inhibition is not obvious. NEW METHOD: In this paper, we propose an in silico interventional procedure that makes predictions about the effect of stimulating or inhibiting single neurons on other neurons, and thereby gives the ability to accurately identify inhibitory effects. COMPARISON: To experimentally test these predictions, we have developed a Neural Circuit Probe (NCP) that delivers drugs transiently and reversibly on individually identified neurons to assess their contributions to the neural circuit behavior. RESULTS: Using the NCP, putative inhibitory connections identified by the in silico procedure were validated through in vitro interventional experiments. CONCLUSIONS: Together, these results demonstrate how detailed microcircuitry can be inferred from statistical models derived from neurophysiology data.

In vivo synaptic activity-independent co-uptakes of amyloid ß1-42 and Zn2+ into dentate granule cells in the normal brain.

Neuronal amyloid ß1-42 (Aß1-42) accumulation is considered an upstream event in Alzheimer's disease pathogenesis. Here we report the mechanism on synaptic activity-independent Aß1-42 uptake in vivo. When Aß1-42 uptake was compared in hippocampal slices after incubating with Aß1-42, In vitro Aß1-42 uptake was preferentially high in the dentate granule cell layer in the hippocampus. Because the rapid uptake of Aß1-42 with extracellular Zn2+ is essential for Aß1-42-induced cognitive decline in vivo, the uptake mechanism was tested in dentate granule cells in association with synaptic activity. In vivo rapid uptake of Aß1-42 was not modified in the dentate granule cell layer after co-injection of Aß1-42 and tetrodotoxin, a Na+ channel blocker, into the dentate gyrus. Both the rapid uptake of Aß1-42 and Zn2+ into the dentate granule cell layer was not modified after co-injection of CNQX, an AMPA receptor antagonist, which blocks extracellular Zn2+ influx, Both the rapid uptake of Aß1-42 and Zn2+ into the dentate granule cell layer was not also modified after either co-injection of chlorpromazine or genistein, an endocytic repressor. The present study suggests that Aß1-42 and Zn2+ are synaptic activity-independently co-taken up into dentate granule cells in the normal brain and the co-uptake is preferential in dentate granule cells in the hippocampus. We propose a hypothesis that Zn-Aß1-42 oligomers formed in the extracellular compartment are directly incorporated into neuronal plasma membranes and form Zn2+-permeable ion channels.

Transtympanic Delivery of Local Anesthetics for Pain in Acute Otitis Media.

Acute otitis media (AOM) commonly causes pain and distress in children. Existing analgesic ototopical drops have limited effectiveness due to the impermeable nature of the tympanic membrane. We developed a local drug delivery system to provide sustained pain relief in patients with AOM, achieved by applying a single dose of a hydrogel formulation onto the tympanic membrane. Successful drug delivery across intact tympanic membranes was demonstrated using the amino-amide anesthetic, bupivacaine, and a highly potent site 1 sodium channel blocker anesthetic, tetrodotoxin. The chemical permeation enhancers incorporated in the delivery system increased the permeability of the tympanic membrane to the anesthetics considerably. The drug levels measured using a previously developed ex vivo model reflect the potential for highly effective local anesthesia.

Two coupled circadian oscillations regulate Bmal1-ELuc and Per2-SLR2 expression in the mouse suprachiasmatic nucleus.

Circadian rhythms in clock genes, Bmal1 and Per2 expression were monitored simultaneously in the cultured slice of mouse suprachiasmatic nucleus (SCN) by dual bioluminescent reporters. In the neonatal SCN, the phase-relation between the Bmal1 and Per2 rhythms were significantly changed during culture. Medium exchange produced phase-dependent phase shifts (PRCm) in the Bmal1 rhythms, but not in the Per2 rhythms. As a result, the two circadian rhythms were temporally dissociated after medium exchange. In the adult SCN, the phase-relation between the two rhythms was kept constant during culture at least up to 20 cycles. The amplitude of PRCm in the adult SCN was significantly attenuated in the Bmal1 rhythm, whereas a PRCm was developed in the Per2 rhythm. The circadian period was not systematically affected by medium exchange in either of rhythms, regardless of whether it was in the neonatal or the adult SCN. Tetrodotoxin, a sodium channel blocker, enhanced the phase-response in both rhythms but abolished the phase-dependency. In addition, tetrodotoxin lengthened the circadian period independent of the phase of administration. Thus, the Bmal1 and Per2 rhythms in the SCN are dissociable and likely regulated by distinct circadian oscillators. Bmal1 is the component of a Bmal1/REV-ERBa/ROR loop and Per2 a Per/Cry/BMAL1/CLOCK loop. Both loops could be molecular mechanisms of the two circadian oscillators that are coupled through the protein product of Bmal1. The coupling strength between the two oscillations depends on developmental stages.

The Acute Toxicity of Tetrodotoxin and Tetrodotoxin⁻Saxitoxin Mixtures to Mice by Various Routes of Administration.

Tetrodotoxin (TTX) is a potent neurotoxin associated with human poisonings through the consumption of pufferfish. More recently, TTX has been identified in bivalve molluscs from diverse geographical environments, including Europe, and is therefore recognised as an emerging threat to food safety. A recent scientific opinion of the EFSA Panel on Contaminants in the Food Chain recognised the need for further data on the acute oral toxicity of TTX and suggested that, since saxitoxin (STX) and TTX had similar modes of action, it was possible that their toxicities were additive so could perhaps be combined to yield one health-based guideline value. The present study determined the toxicity of TTX by various routes of administration. The testing of three different mixtures of STX and TTX and comparing the experimentally determined values to those predicted on the basis of additive toxicity demonstrated that the toxicities of STX and TTX are additive. This illustrates that it is appropriate to treat TTX as a member of the paralytic shellfish group of toxins. Since the toxicity of TTX was found to be the same as STX by feeding, a molar toxicity equivalence factor of 1.0 for TTX can be applied.

Graded Transmission without Action Potentials Sustains Rhythmic Activity in Some But Not All Modulators That Activate the Same Current.

Neurons in the central pattern-generating circuits in the crustacean stomatogastric ganglion (STG) release neurotransmitter both as a graded function of presynaptic membrane potential that persists in TTX and in response to action potentials. In the STG of the male crab Cancer borealis, the modulators oxotremorine, C. borealis tachykinin-related peptide Ia (CabTRP1a), red pigment concentrating hormone (RPCH), proctolin, TNRNFLRFamide, and crustacean cardioactive peptide (CCAP) produce and sustain robust pyloric rhythms by activating the same modulatory current (IMI), albeit on different subsets of pyloric network targets. The muscarinic agonist oxotremorine, and the peptides CabTRP1a and RPCH elicited rhythmic triphasic intracellular alternating fluctuations of activity in the presence of TTX. Intracellular waveforms of pyloric neurons in oxotremorine and CabTRP1a in TTX were similar to those in the intact rhythm, and phase relationships among neurons were conserved. Although cycle frequency was conserved in oxotremorine and TTX, it was altered in CabTRP1a in the presence of TTX. Both rhythms were primarily driven by the pacemaker kernel consisting of the Anterior Burster and Pyloric Dilator neurons. In contrast, in TTX the circuit remained silent in proctolin, TNRNFLRFamide, and CCAP. These experiments show that graded synaptic transmission in the absence of voltage-gated Na+ current is sufficient to sustain rhythmic motor activity in some, but not other, modulatory conditions, even when each modulator activates the same ionic current. This further demonstrates that similar rhythmic motor patterns can be produced by qualitatively different mechanisms, one that depends on the activity of voltage-gated Na+ channels, and one that can persist in their absence.SIGNIFICANCE STATEMENT The pyloric rhythm of the crab stomatogastric ganglion depends both on spike-mediated and graded synaptic transmission. We activate the pyloric rhythm with a wide variety of different neuromodulators, all of which converge on the same voltage-dependent inward current. Interestingly, when action potentials and spike-mediated transmission are blocked using TTX, we find that the muscarinic agonist oxotremorine and the neuropeptide CabTRP1a sustain rhythmic alternations and appropriate phases of activity in the absence of action potentials. In contrast, TTX blocks rhythmic activity in the presence of other modulators. This demonstrates fundamental differences in the burst-generation mechanisms in different modulators that would not be suspected on the basis of their cellular actions at the level of the targeted current.

Pharmacological characterization of crotamine effects on mice hind limb paralysis employing both ex vivo and in vivo assays: Insights into the involvement of voltage-gated ion channels in the crotamine action on skeletal muscles.

The high medical importance of Crotalus snakes is unquestionable, as this genus is the second in frequency of ophidian accidents in many countries, including Brazil. With a relative less complex composition compared to other genera venoms, as those from the Bothrops genus, the Crotalus genus venom from South America is composed basically by the neurotoxin crotoxin (a phospholipase A2), the thrombin-like gyroxin (a serinoprotease), a very potent aggregating protein convulxin, and a myotoxic polypeptide named crotamine. Interestingly not all Crotalus snakes express crotamine, which was first described in early 50s due to its ability to immobilize animal hind limbs, contributing therefore to the physical immobilization of preys and representing an important advantage for the envenoming efficacy, and consequently, for the feeding and survival of these snakes in nature. Representing about 10-25% of the dry weight of the crude venom of crotamine-positive rattlesnakes, the polypeptide crotamine is also suggested to be of importance for antivenom therapy, although the contribution of this toxin to the main symptoms of envenoming process remains far unknown until now. Herein, we concomitantly performed in vitro and in vivo assays to show for the first time the dose-dependent response of crotamine-triggered hind limbs paralysis syndrome, up to now believed to be observable only at high (sub-lethal) concentrations of crotamine. In addition, ex vivo assay performed with isolated skeletal muscles allowed us to suggest here that compounds active on voltage-sensitive sodium and/or potassium ion channels could both affect the positive inotropic effect elicited by crotamine in isolated diaphragm, besides also affecting the hind limbs paralysis syndrome imposed by crotamine in vivo. By identifying the potential molecular targets of this toxin, our data may contribute to open new roads for translational studies aiming to improve the snakebite envenoming treatment in human. Interestingly, we also demonstrate that the intraplantal or intraperitoneal (ip) injections of crotamine in mice do not promote pain. Therefore, this work may also suggest the profitable utility of non-toxic analogs of crotamine as a potential tool for targeting voltage-gated ion channels in skeletal muscles, aiming its potential use in the therapy of neuromuscular dysfunctions and envenoming therapy.

Impaired neural pathway in gastric muscles of patients with diabetes.

To explore the pathogenic mechanism of diabetic gastropathy, we investigated differences in response to electrical field stimulation (EFS) of gastric muscles from diabetic and non-diabetic (control) patients. Gastric specimens were obtained from 34 patients and 45 controls who underwent gastrectomy for early gastric cancer. Using organ bath techniques, we examined peak and nadir values of contraction under EFS. To examine responses to purinergic and nitrergic inhibition without cholinergic innervation, atropine, MRS2500, and N-nitro-L-arginine (L-NNA) were added sequentially to the organ bath. Tetrodotoxin (TTX) was used to confirm that the responses to EFS were mediated via neural stimulation. In the absence of pharmacological agents, peak contraction amplitude was greater in non-diabetic controls compared to diabetics only in the distal longitudinal gastric muscles. However, the nadir was greater in controls than in patients in both proximal and distal gastric circular muscles. Addition of MRS2500 could not decrease the nadir in both controls and patients, both in the proximal and distal stomach. However, L-NNA completely reversed the relaxation. TTX had no further effect on nadir. In conclusion, impaired inhibitory nitrergic neural pathway in both proximal and distal stomach and impaired excitatory cholinergic neural pathway in the distal stomach may contribute to the pathogenic mechanism underlying diabetic gastropathy.

Hollow Silica Nanoparticles Penetrate the Peripheral Nerve and Enhance the Nerve Blockade from Tetrodotoxin.

The efficacy of tetrodotoxin (TTX), a very potent local anesthetic, is limited by its poor penetration through barriers to axonal surfaces. To address this issue, we encapsulated TTX in hollow silica nanoparticles (TTX-HSN) and injected them at the sciatic nerve in rats. TTX-HSN achieved an increased frequency of successful blocks, prolonged the duration of the block, and decreased the toxicity compared to free TTX. In animals injected with fluorescently labeled HSN, the imaging of frozen sections of nerve demonstrated that HSN could penetrate into nerve and that the penetrating ability of silica nanoparticles was highly size-dependent. These results demonstrated that HSN could deliver TTX into the nerve, enhancing efficacy while improving safety.

Enhanced Triggering of Local Anesthetic Particles by Photosensitization and Photothermal Effect Using a Common Wavelength.

On-demand pain relief systems would be very helpful additions to the armamentarium of pain management. Near-infrared triggered drug delivery systems have demonstrated the potential to provide such care. However, challenges remain in making such systems as stimulus-sensitive as possible, to enhance depth of tissue penetration, repeatability of triggering, and safety. Here we developed liposomes containing the local anesthetic tetrodotoxin and also containing a photosensitizer and gold nanorods that were excitable at the same near-infrared wavelength. The combination of triggering mechanisms enhanced the photosensitivity and repeatability of the system in vitro when compared with liposomes with a single photoresponsive component. In vivo, on-demand local anesthesia could be induced with a low irradiance and short irradiation duration, and liposomes containing both photosensitizer and gold nanorods were more effective than those containing just one photoresponsive component. Tissue reaction was benign.